U.S. patent number 6,576,436 [Application Number 09/457,476] was granted by the patent office on 2003-06-10 for anticancer agents based on prevention of protein prenylation.
This patent grant is currently assigned to The Arizona Disease Control Research Commission. Invention is credited to Rosemarie Hartman, Scott Lefler, Karl J. Okolotowicz, Steven R. Ottersberg, Dale E. Robinson, Seth D. Rose.
United States Patent |
6,576,436 |
Rose , et al. |
June 10, 2003 |
**Please see images for:
( Certificate of Correction ) ** |
Anticancer agents based on prevention of protein prenylation
Abstract
Prenylating enzymes are involved in modifying oncoproteins, such
as RAS, so that growth of neoplastic cells becomes uncontrolled.
Inactivation of such enzymes can prevent uncontrolled growth.
.alpha.-Dicarbonyl compounds can be used to covalently modify and
thereby inactivate prenylating enzymes such as protein
farnesyltransferase and protein geranylgeranyltransferase. The
compounds can be designed to enhance affinity and/or specificity
for a particular protein substrate.
Inventors: |
Rose; Seth D. (Tempe, AZ),
Ottersberg; Steven R. (Tempe, AZ), Okolotowicz; Karl J.
(Tempe, AZ), Robinson; Dale E. (San Diego, CA), Hartman;
Rosemarie (Tempe, AZ), Lefler; Scott (Tempe, AZ) |
Assignee: |
The Arizona Disease Control
Research Commission (Phoenix, AZ)
|
Family
ID: |
22338782 |
Appl.
No.: |
09/457,476 |
Filed: |
December 9, 1999 |
Current U.S.
Class: |
435/15;
514/561 |
Current CPC
Class: |
C12N
9/99 (20130101); A61P 35/00 (20180101); A61K
31/121 (20130101); C12N 9/1085 (20130101); A61K
31/12 (20130101); C12Q 1/48 (20130101); C12Y
205/01029 (20130101); A61K 31/195 (20130101); G01N
2500/04 (20130101) |
Current International
Class: |
A61K
31/12 (20060101); A61K 31/121 (20060101); C12Q
1/48 (20060101); C12N 9/10 (20060101); C12Q
001/48 () |
Field of
Search: |
;435/15,29 ;514/561 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Pande et al. (1980). Camphorquinone-10-sulfonic acid and
derivatives: Convenient reagents for reversible modification of
arginine residues. Proc. Natl. Acad. Sci. USA 77(2), pp 895-899.*
.
CAPLUS abstract (Acc No 1970:520240). Schank et al. (1970).
Alpha-hydroxy-beta-oxosulfones: Influence of conjugation and
substitution effects. Chem.Ber. 103(10): pp 3087-3092.* .
Mori et al. (1991). Preparative bioorganic chemistry. Synthesis 6:
pp 487-490.* .
Neidlein et al. (1996). Syntheses of 2,3-dioxoalkylphosphonates and
otehr novel beta-ketophosphonates as well as of a
phosphoinopyruvamide. Helv. Chim. Acta 79(3): pp 895-912.* .
Wedzicha et al. (1975). Concentrations of sulfonates derived from
sulphite in certain foods. J. Sci. Fd. Agric. 26: pp 327-335.*
.
Sohn et al. "Chemical Modification Studies of Yeast Farnesyl
Protein Transferase" J. Biochem Mol. Biol. vol. 30, No. 4, pp.
280-284, Jul. 31, 1997. .
Barnard et al. "Characterization of Liver Prenyl Transferase and
its Inactivation by Phenylglyoxal Biochimica et Biophysica Acta."
Feb. 22, 1980, vol. 617, No. 2, pp. 169-182. .
Barnard et al. "Human Liver Prenyltransferase and its
Characterization" Biochimcia et Biophysica Acta. Sep. 15, 1981,
vol. 661, No. 1, pp. 87-99. .
Boivin et al. "Essential Arginine Residues in Isoprenylcysteine
Protein Carboxyl Methyltransferase" Biochem. Cell Biol. 1977, vol.
75, No. 1, pp. 63-69..
|
Primary Examiner: Gitomer; Ralph
Attorney, Agent or Firm: Banner & Witcoff, Ltd.
Parent Case Text
This application claims the benefit of provisional application Ser.
No. 60/111,478 filed Dec. 9, 1998. The disclosure of the
provisional application is expressly incorporated by reference
herein.
Claims
We claim:
1. A method of inactivating a prenylation enzyme, comprising the
step of: contacting a prenylation enzyme, in the presence of at
least one of its natural substrates, with an .alpha.-dicarbonyl
compound having a formula:
(Formula III) wherein A is an anionic group selected from the group
consisting of carboxylate, sulfate, sulfonate, sulfinate,
phosphate, phosphonate, thiophosphate, pyrophosphate, enolate,
sulfonamide, sulfone, and tetrazole, or a group that forms an
anionic group selected from the group consisting of carboxylate,
sulfate, sulfonate, sulfinate, phosphate, phosphonate,
thiophosphate, pyrophosphate, enolate, sulfonamide, sulfone, and
tetrazole at physiological pH; wherein L is selected from the group
consisting of alkyl, alkenyl, alkynyl, and aryl; wherein R is
selected from the group consisting of hydrogen, alkyl, alkenyl,
alkynyl, and aryl; and wherein D is a dicarbonyl functional group
or a masked form of said group, whereby the activity of the
prenylation enzyme is reduced by at least 50%.
2. A method of screening compounds as anti-tumor agents comprising:
contacting a test compound with a prenylation enzyme, wherein the
test compound has a formula:
(Formula III) wherein A is an anionic group selected from the group
consisting of carboxylate, sulfate, sulfonate, sulfinate,
phosphate, phosphonate, thiophosphate, pyrophosphate, enolate,
sulfonamide, sulfone, and tetrazole, or a group that forms an
anionic group selected from the group consisting of carboxylate,
sulfate, sulfonate, sulfinate, phosphate, phosphonate,
thiophosphate, pyrophosphate, enolate, sulfonamide, sulfone, and
tetrazole at physiological pH; wherein L is selected from the group
consisting of alkyl, alkenyl, alkynyl, and aryl; wherein R is
selected from the group consisting of hydrogen, alkyl, alkenyl,
alkynyl, and aryl; and wherein D is a dicarbonyl functional group
or a masked form of said group, measuring prenylation activity of
the enzyme, wherein a test compound which reduces prenylation
activity at least 50% is a candidate anti-tumor agent.
3. A method of inhibiting the growth of a cancer cell, comprising
the step of: contacting a cancer cell with an .alpha.-dicarbonyl
compound having a formula:
(Formula III) wherein A is an anionic group selected from the group
consisting of carboxylate, sulfate, sulfonate, sulfinate,
phosphate, phosphonate, thiophosphate, pyrophosphate, enolate,
sulfonamide, sulfone, and tetrazole, or a group that forms an
anionic group selected from the group consisting of carboxylate,
sulfate, sulfonate, sulfinate, phosphate, phosphonate,
thiophosphate, pyrophosphate, enolate, sulfonamide, sulfone, and
tetrazole at physiological pH; wherein L is selected from the group
consisting of alkyl, alkenyl, alkynyl, and aryl; wherein R is
selected from the group consisting of hydrogen, alkyl, alkenyl,
alkynyl, and aryl; and wherein D is a dicarbonyl functional group
or a masked form of said group, whereby the growth of the cancer
cell is inhibited.
4. A pharmaceutically acceptable formulation comprising: a compound
having a formula:
(Formula III) wherein A is an anionic group selected from the group
consisting of carboxylate, sulfate, sulfonate, sulfinate,
phosphate, phosphonate, thiophosphate, pyrophosphate, enolate,
sulfonamide, sulfone, and tetrazole, or a group that forms an
anionic group selected from the group consisting of carboxylate,
sulfate, sulfonate, sulfinate, phosphate, phosphonate,
thiophosphate, pyrophosphate, enolate, sulfonamide, sulfone, and
tetrazole at physiological pH; wherein L is selected from the group
consisting of alkyl, alkenyl, alkynyl, and aryl; wherein R is
selected from the group consisting of hydrogen, alkyl, alkenyl,
alkynyl, and aryl; and wherein D is a dicarbonyl functional group
or a masked form of said group, and a pharmaceutically acceptable
excipient.
Description
TECHNICAL FIELD OF THE INVENTION
This invention relates to the use of molecules with adjacent
carbonyl groups (O.dbd.C--C.dbd.O), or related functional groups,
that inactivate one or more prenyltransferases in cancer cells and
thereby prevent the unrestrained division of cancer cells.
BACKGROUND OF THE INVENTION
More than half of all human cancers contain a mutant gene that
encodes a mutant protein known as Ras. In cancer cells, Ras
activates the cells to divide in an unrestrained manner. To induce
cell division, Ras must be localized at the inner surface of the
cancer cell membrane. Membrane localization of Ras is effected by
attachment of a hydrophobic group, typically the farnesyl group,
which becomes attached to Ras. In some cells, the related
geranylgeranyl group becomes attached instead. Both of these groups
become attached to Ras enzymatically, in a process known as
prenylation. Thus, interference with prenylation of Ras has the
potential to prevent Ras localization at the inner surface of the
cancer cell membrane, resulting in the cessation of unrestrained
cell division of the cancer cell.
The enzyme that attaches the farnesyl group to Ras protein to
facilitate the latter's localization at the inner surface of the
cancer cell membrane is farnesyl protein transferase, also known as
protein farnesyltransferase (herein referred to as FTase). The
famesyl group becomes attached to Ras by reaction with farnesyl
diphosphate, a natural FTase substrate, also known as farmesyl
pyrophosphate (herein referred to as FPP). In other words, FTase
catalyzes the following reaction, in which Ras becomes attached to
the farnesyl group by displacement of pyrophosphate (P.sub.2
O.sub.7.sup.4-, herein referred to as PP.sub.i): ##STR1##
The newly formed farnesyl-Ras localizes at the inner surface of the
cancer cell membrane and causes the cancer cell to divide without
restraint.
There is a continuing need in the art for new ways to inhibit the
growth of cancer cells. There is a need in the art for new ways to
inactivate targets which are specifically involved in cancer
progression and development.
SUMMARY OF INVENTION
It is an object of the invention to provide methods for
inactivating prenylating enzymes.
It is another object of the invention to provide methods for
screening test compounds for the ability to inactivate prenylating
enzymes.
It is yet another object of the invention to provide methods for
inhibiting the growth of a cancer cell.
It is still another object to provide pharmaceutical compositions
for treating cancer.
It is another object to provide new compounds useful for treating
cancers or inhibiting enzymes.
These and other objects of the invention are provided by one or
more of the embodiments described below. In one embodiment a method
of inactivating a prenylation enzyme is provided. The method
comprises the step of contacting a prenylation enzyme with an
.alpha.-dicarbonyl compound having formula (I):
R1-(C.dbd.O)--(C.dbd.O)--R2, wherein R1 is selected from the group
consisting of hydrogen, alky, alkenyl, alkynyl, and aryl; and
wherein R2 is selected from the group consisting of hydrogen,
alkyl, alkenyl, alkynyl, and aryl; and whereby the activity of the
prenylation enzyme is reduced by at least 50%.
According to another embodiment of the invention, a method of
screening compounds as anti-tumor agents is provided. The method
comprises the step of contacting a test compound with a prenylation
enzyme. The test compound has formula (I):
R1-(C.dbd.O)--(C.dbd.O)--R2, wherein R1 is selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, and aryl; and R2
is selected from the group consisting of hydrogen, alkyl, alkenyl,
alkynyl, and aryl. Prenylation activity of the enzyme is measured.
A test compound which reduces prenylation activity at least 50% is
identified as a candidate anti-tumor agent.
According to another aspect of the invention a method of
inactivating a prenylation enzyme is provided. The method comprises
the step of contacting a prenylation enzyme with an
.alpha.-dicarbonyl compound having formula (II):
R1-(C.dbd.O)--(C.dbd.O)--L--(C.dbd.O)--(C.dbd.O)--R2, wherein R1,
R2, and L are independently selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, and aryl. The activity of the
prenylation enzyme is reduced by at least 50%.
According to another aspect of the invention a method of screening
compounds as anti-tumor agents is provided. A test compound is
contacted with a prenylation enzyme. The test compound has formula
(II): R1-(C.dbd.O)--(C.dbd.O)--L--(C.dbd.O)--(C.dbd.O)--R2, wherein
R1, R2, and L are independently selected from the group consisting
of hydrogen, alkyl, alkenyl, alkynyl, and aryl. Prenylation
activity of the enzyme is measured. A test compound which reduces
prenylation activity at least 50% is a candidate anti-tumor
agent.
Another embodiment of the invention provides yet another method of
inactivating a prenylation enzyme. A prenylation enzyme is
contacted with an .alpha.-dicarbonyl compound having formula (III):
A--L--D--R, wherein A is selected from the group consisting of an
anionic group, a group that can spontaneously become anionic at
physiological pH, and a group that can enzymatically become anionic
at physiological pH; wherein L is a linker group; wherein D is a
dicarbonyl functional group or a masked form of said group, and
wherein R is selected from the group consisting of hydrogen, alkyl,
alkenyl, alkynyl, and aryl. The activity of the prenylation enzyme
is reduced by at least 50%.
Still another aspect of the invention is a method of screening
compounds as anti-tumor agents. A test compound is contacted with a
prenylation enzyme. The test compound has formula (III):
A--L--D--R, wherein A is selected from the group consisting of an
anionic group, a group that can spontaneously become anionic at
physiological pH, and a group that can enzymatically become anionic
at physiological pH; wherein L is a linker group; R is selected
from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, and
aryl, and wherein D is a dicarbonyl functional group or a masked
form of said group. Activity of the prenylation enzyme is measured.
A test compound which reduces prenylation activity at least 50% is
a candidate anti-tumor agent.
Also provided as an embodiment of the invention is a method of
inhibiting growth of cancer cells. A cancer cell is contacted with
an .alpha.-dicarbonyl compound having a formula:
R1-(C.dbd.O)--(C.dbd.O)--R2. R1 is selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, and aryl. R2 is
selected from the group consisting of hydrogen, alkyl, alkenyl,
alkynyl, and aryl. The growth of the cancer cell is consequently
inhibited.
Another aspect of the invention is a method of inhibiting the
growth of a cancer cell in which a cancer cell is contacted with an
.alpha.-dicarbonyl compound having a formula:
R1-(C.dbd.O)--(C.dbd.O)--L--(C.dbd.O)--(C.dbd.O)--R2. R1, L, and R2
are independently selected from the group consisting of hydrogen,
alkyl, alkenyl, alkynyl, and aryl. The growth of the cancer cell is
consequently inhibited.
Another embodiment of the invention is a method of inhibiting the
growth of a cancer cell. A cancer cell is contacted with an
.alpha.-dicarbonyl compound having a formula: A--L--D--R. A is
selected from the group consisting of an anionic group, a group
that can spontaneously become anionic at physiological pH, and a
group that can enzymatically become anionic at physiological pH. L
is a linker group, and R is selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, and aryl. D is a dicarbonyl
functional group or a masked form of said group. The growth of the
cancer cell is consequently inhibited.
Also provided by the present invention is a pharmaceutically
acceptable formulation. The formulation comprises a compound
according to Formula I, Formula II, or Formula III, and a
pharmaceutically acceptable excipient.
Additionally provided by the present invention is a compound
according to Formula I, Formula II, or Formula III. The compound is
not phenylglyoxal, biphenyldiglyoxaldehyde, 2-oxododecanal, or
2,3-pentanedione.
The present invention thus provides a method for hampering or
preventing the proliferation of cancer cells, resulting in a
decrease in tumor size and/or disappearance of the cancer. It acts
by interference with cancer cell biochemistry, in which the enzyme
famesyl protein transferase, geranylgeranyl protein transferase,
and/or another prenylation enzyme acts on oncogenic proteins, such
as RAS, or other growth-related cellular protein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Assay of FTase after incubation of the enzyme in the
absence (upper curve) and presence (lower curve) of inactivator (10
mM PGO for 10 min). Activity of human recombinant FTase was
monitored by the increase in fluorescence at .lambda.=505 nm, with
farnesyl pyrophosphate and Ds-GCVLS (as a substitute for Ras) as
substrates, after removal of PGO with a high molecular weight
cutoff microconcentrator. The lower curve shows the virtual absence
of enzyme activity following preincubation of FTase with PGO. Each
point is the average of duplicate determinations.
FIG. 2. Kinetics of inactivation of FTase by PGO. The rate of
inactivation of FTase is dependent on PGO concentration. Loss of
enzyme activity is faster at higher concentrations of PGO:
(.box-solid.) PGO absent, (.diamond-solid.) 5 mM PGO, and (.DELTA.)
10 mM PGO. At 20 mM PGO, enzyme activity was lost in 2 min of
incubation (data not shown). Assays were carried out after removal
of PGO with a high molecular weight cutoff microconcentrator. Each
point is the average of duplicate determinations.
FIG. 3. Farnesyl pyrophosphate protection of FTase from
inactivation by PGO. Points are determined by 100.times.(FTase
activity after incubation with FPP and PGO)/(FTase activity after
incubation with FPP without PGO). Assays were carried out after
removal of FPP and PGO with a high molecular weight cutoff
microconcentrator. Each point is the average of duplicate
determinations.
FIG. 4. Kinetics of inactivation of FTase by 2-oxododecanal. The
rate of inactivation of FTase is dependent on 2-oxododecanal
concentration. Loss of enzyme activity is faster at higher
concentrations of 2-oxododecanal: (.box-solid.) 2-oxododecanal
absent, (.diamond-solid.) 5 mM 2-oxododecanal, and (.DELTA.) 10 mM
2-oxododecanal. At 35 mM 2-oxododecanal, enzyme activity was lost
in 2 min of incubation (data not shown). Dimethyl sulfoxide was
present at 5% in the incubation mixtures (including the control).
Assays were carried out after removal of 2-oxododecanal with a high
molecular weight cutoff microconcentrator. Each point is the
average of duplicate determinations.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Key targets in a strategy to retard cancer cell proliferation are
the prenylation enzymes, such as the enzyme FTase. By reducing or
destroying FTase activity, we have found that we can prevent Ras
farnesylation, which in turn prevents localization of Ras at the
inner surface of the cancer cell membrane, thereby preventing Ras
from causing the cancer cells to divide and proliferate.
Many substances are known to block FTase activity and prevent
farnesylation of Ras. They consist of inhibitors of the enzyme
FTase, and they generally operate by blocking the binding of Ras or
FPP (or both) to FTase. Without the normal substrates (Ras and FPP)
being able to bind to FTase, FTase cannot transfer the farnesyl
group from FPP to Ras. Inhibitors structurally mimic one or both of
the natural substrates of the enzyme, Ras and/or FPP. Their binding
is noncovalent and reversible. In other words, the binding of the
inhibitor to FTase does not involve the formation of covalent bonds
between the inhibitor and FTase. Instead, hydrophobic forces,
hydrogen bonding, electrostatic attraction, etc. are the forces
principally responsible for binding of the inhibitor to the enzyme
FTase. These binding forces allow the inhibitor to block the site
on FTase where the normal substrates need to bind for farnesylation
of Ras to take place
We have found that a potentially more useful method of preventing
FTase from farnesylating Ras can be achieved, namely inactivation
of FTase. Interaction of FTase with substances that are
inactivators results in the covalent attachment of a part or all of
the inactivator to FTase. In principle, the covalent attachment can
be irreversible or nearly irreversible. Also, the specificity of
the inhibitor for the target enzyme FTase can be more finely tuned
because it is based on two parameters: (1) structural similarity to
the normal substrates (FPP and/or Ras); (2) a reactivity that is
appropriate for the chemical groups that are a part of the enzyme
FTase, particularly those that participate in the actual process
catalyzed by FTase (i.e., those that more-or-less directly
participate in the transfer of the farnesyl group of FPP to
Ras).
The fact that FPP loses pyrophosphate, which is anionic, might be
indicative of the presence of a structural feature in FTase that
would assist in the displacement of pyrophosphate, for example by
ionic bonding to a positively charged group in the enzyme's active
site. One such possibility is the side chain of arginine
[R--NH.dbd.C(NH.sub.2).sub.2.sup.+ ]. Indeed, the X-ray
crystallographic structure determination of FTase reveals the
presence of arginine residues in the vicinity of the hypothetical
binding site of FPP, which further suggests the possible role of
arginine in assisting with the removal of pyrophosphate as the
farnesyl group becomes bonded to Ras.
Some chemical reagents that react with the side chain of arginine
residues in proteins possess a functional group that comprises at
least two adjacent carbonyl groups, i.e., O.dbd.C--C.dbd.O. Such a
functional group binds covalently to the nitrogens of the side
chain of arginine, causing the vicinity of the arginine to become
blocked and possibly the positive charge to be lost. Hypothetical
structures of the adduct(s) of the arginine side chain and the
dicarbonyl compound phenylglyoxal are shown below: ##STR2##
Our invention consists of chemical agents bearing at least two
adjacent carbonyl groups (O.dbd.C--C.dbd.O) designed to react
(i.e., covalently interact) with the arginine side chain.
Furthermore, to target better the substance to FTase in preference
to other enzymes, and to target better the substance to the active
site of FTase in preference to the external or noncatalytic portion
of the enzyme, a preferred embodiment of the invention consists of
a substance with at least one dicarbonyl functional group and other
structural features which favor the binding of the agent to the
active site of FTase. For example, the active site of FTase is
hydrophobic to bind the natural substrate FPP which contains a
hydrophobic region (i.e., the farnesyl group). A hydrophobic
substance that also contains the dicarbonyl functional group is
preferred, a class represented by the following generalized
structural formula: ##STR3##
wherein R and R' represent hydrogen and/or a hydrophobic group,
such as an alkyl, alkenyl, alkynyl, and aryl group. The group can
be cyclic or noncyclic, branched or unbranched, with or without
unsaturation (e.g., aryl, alkenyl) or substituents. Preferably the
group is designed to impart specificity of the substance for
binding to and/or inactivation of FTase.
The term "alkyl," as used alone or in combination herein, refers to
an unsubstituted or optionally substituted, straight, or branched
chain saturated hydrocarbon group containing from one to
twenty-five carbon atoms, preferably from one to fifteen carbons,
such as methyl, ethyl, n-propyl, n-butyl, pentyl, hexyl, heptyl,
octyl the various branch chain isomers thereof, such as isopropyl,
isobutyl, sec-butyl, tert-butyl, isohexyl and the like. The alkyl
group may be optionally substituted by one or more substituents,
and generally no more than three, and most often just one
substituent. Preferred optional substituents include halo, alkoxy,
amino, mono- and di-substituted amino, aryl, carboxylic acid,
heterocyclo, heteroaryl, cycloalkyl, hydroxy, trifluoromethoxy and
the like. The term "lower alkyl" refers to such alkyl groups
containing from one to five carbon atoms.
The term "alkoxy," as used alone or in combination herein, refers
to an alkyl group, as defined above, covalently bonded to the
parent molecule through an --O-- linkage, such as methoxy, ethoxy,
propoxy, isopropoxy, butoxy, t-butoxy and the like.
The term "alkoxyalkyl" refers specifically to an alkyl group
substituted with an alkoxy group.
The term "aryloxy," as used alone or in combination herein, refers
to an aryl group, as defined below, covalently bonded to the parent
molecule through an --O-- linkage. An example of an aryloxy is
phenoxy.
The term "cycloalkoxy," as used alone or in combination herein,
refers to a cycloalkyl group, as defined below, covalently bonded
to the parent molecule through an --O-- linkage.
The term "alkylthio," as used alone or in combination herein,
refers to an alkyl group, as defined above, covalently bonded to
the parent molecule through an --S-- linkage.
The term "alkenyl," as used alone or in combination herein, refers
to an alkyl group, as defined above, containing one or more
carbon-carbon double bonds, preferably one or two double bonds.
Examples of alkenyl include ethenyl, propenyl, 1,3-butadienyl, and
1,3,5-hexatrienyl.
The term "alkynyl," as used alone or in combination herein, refers
to an alkyl group, as defined above, containing one or more
carbon-carbon triple bonds, preferably one or two triple bonds.
The term "cycloalkyl," as used alone or in combination herein,
refers to an unsubstituted or optionally substituted, saturated
cyclic hydrocarbon group containing three to eight carbon atoms.
The cycloalkyl group may optionally be substituted by one or more
substituents, and generally no more than three, and most often just
one substituent. Preferred optional substituents include alkyl,
halo, amino, mono- and di-substituted amino, aryl, hydroxy and the
like.
The term "haloalkyl" is a species of alkyl as defined herein, and
particularly refers to an alkyl, preferably a lower alkyl,
substituted with one or more halogen atoms, and preferably is a
C.sub.1 to C.sub.4 alkyl substituted with one to three halogen
atoms. One example of a haloalkyl is trifluoromethyl.
The term "alkanoyl" as used alone or in combination herein refers
to an acyl radical derived from an alkanecarboxylic acid
(alkyl-C(O)--), particularly a lower alkanecarboxylic acid, and
includes such examples as acetyl, propionyl, butyryl, valeryl, and
4-methylvaleryl.
The term "aroyl" means an acyl radical derived from an aromatic
carboxylic acid, such as optionally substituted benzoic or
naphthoic acids and specifically including benzoyl and
1-naphthoyl.
The term "aminocarbonyl" means an amino-substituted carbonyl
(carbamoyl or carboxamide) wherein the amino group is a primary
amino (--NH.sub.2). Substituted aminocarbonyl refers to secondary
(mono-substituted amino) or tertiary amino (di-substituted amino)
group, as defined below, preferably having as a substituent(s) a
lower alkyl group.
The term "aminoalkanoyl" means an amino-substituted alkanoyl
wherein the amino group is a primary amino group
(-alkyl-C(O)--NH.sub.2). The term "substituted aminoalkanoyl"
refers to related secondary (mono-substituted amino) or tertiary
amino (di-substituted amino) group, as defined below.
The term "carbocycloalkyl" when used alone or in combination refers
to an unsubstituted or optionally substituted, stable, saturated or
partially unsaturated monocyclic, bridged monocyclic, bicyclic, and
spiro ring carbocycles of 3 to 15 carbon atoms such as cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclohexyl,
bicyclooctyl, bicyclononyl, spirononyl and spirodecyl. Cycloalkyls
are thus one specific subset of carbocycloalkyls. The term
"optionally substituted" as it refers to "carbocycloalkyl" herein
indicates that the carbocycloalkyl group may be substituted at one
or more substitutable ring positions by one or more groups
independently selected from alkyl (preferably lower alkyl), alkoxy
(preferably lower alkoxy), nitro, monoalkylamino (preferably a
lower alkylamino), dialkylamino (preferably a di[lower]alkylamino),
cyano, halo, haloalkyl (preferably trifluoromethyl), alkanoyl,
aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl,
alkylamido (preferably lower alkylamido), alkoxyalkyl (preferably a
lower alkoxy[lower]alkyl), alkoxycarbonyl (preferably a lower
alkoxycarbonyl), alkylcarbonyloxy (preferably a lower
alkylcarbonyloxy) and aryl (preferably phenyl), said aryl being
optionally substituted by halo, lower alkyl and lower alkoxy
groups. Generally, there is no more than one optional
substituent.
The term "heterocyclo" as used, alone or in combination, herein
refers to an unsubstituted or optionally substituted, stable,
saturated, or partially unsaturated, monocyclic, bridged
monocyclic, bicyclic, and spiro ring system containing carbon atoms
and other atoms selected from nitrogen, sulfur and/or oxygen.
Preferably, a heterocyclo group is a 5 or 6-membered monocyclic
ring or an 8-11 membered bicyclic ring which consists of carbon
atoms and contains one, two, or three heteroatoms selected from
nitrogen, oxygen and/or sulfur. Heterocyclo includes benz-fused
monocyclic cycloalkyl groups having at least one such heteroatom.
The term "optionally substituted" as it refers to "heterocyclo"
herein indicates that the heterocyclo group may be substituted at
one or more substitutable ring positions by one or more groups
independently selected from alkyl (preferably lower alkyl and
including haloalkyl (preferably trifluoromethyl)), alkoxy
(preferably lower alkoxy), nitro, monoalkylamino (preferably a
lower alkylamino), dialkylamino (preferably a di[lower]alkylamino),
cyano, halo, alkanoyl, aminocarbonyl, monoalkylaminocarbonyl,
dialkylaminocarbonyl, alkylamido (preferably lower alkylamido),
alkoxyalkyl (preferably a lower alkoxy[lower]alkyl), alkoxycarbonyl
(preferably a lower alkoxycarbonyl), alkylcarbonyloxy (preferably a
lower alkylcarbonyloxy) and aryl (preferably phenyl), said aryl
being optionally substituted by halo, lower alkyl and lower alkoxy
groups. Generally, there is no more than one optional substituent.
Several non-limiting examples of such heterocyclo groups are
illustrated below: ##STR4##
The heterocyclo group may be, and generally is attached to the
parent structure through a carbon atom, or alternatively may be
attached through any heteroatom of the heterocyclo group that
results in a stable structure.
The term "heteroaryl" as used alone or in combination, herein
refers to an unsubstituted or optionally substituted, stable,
aromatic monocyclic or bicyclic ring system containing carbon atoms
and other atoms selected from nitrogen, sulfur and/or oxygen.
Preferably, a heteroaryl group is a 5- or 6-membered monocyclic
ring (optionally benzofused) or an 8-11 membered bicyclic ring
which consists of carbon atoms and contains one, two, or three
heteroatoms selected from nitrogen, oxygen and/or sulfur. The term
"optionally substituted" as it refers to "heteroaryl" herein
indicates that the heteroaryl group may be substituted at one or
more substitutable ring positions by one or more groups
independently selected from alkyl (preferably lower alkyl and
including haloalkyl (preferably trifluoromethyl)), alkoxy
(preferably lower alkoxy), nitro, monoalkylamino (preferably a
lower alkylamino), dialkylamino (preferably a di[lower]alkylamino,
cyano, halo, alkanoyl, aminocarbonyl, monoalkylaminocarbonyl,
dialkylaminocarbonyl, alkylamido (preferably lower alkylamido),
alkoxyalkyl (preferably a lower alkoxy[lower]alkyl), alkoxycarbonyl
(preferably a lower alkoxycarbonyl), alkylcarbonyloxy (preferably a
lower alkylcarbonyloxy) and aryl (preferably phenyl), said aryl
being optionally substituted by halo, lower alkyl and lower alkoxy
groups. Generally, there is no more than one optional substituent.
Several non-limiting examples of such heteroaryl groups are
illustrated below: ##STR5##
The heteroaryl group may be, and generally is attached to the
parent structure through a carbon atom or alternatively may be
attached through any heteroatom of the heteroaryl group that
results in a stable structure. In the foregoing structures it also
is contemplated that a nitrogen could be replaced with an
N-oxide.
Both heterocyclo and heteroaryl also are intended to embrace benzo
fused structures such as 1,2-methylenedioxybenzene and
1,4-benzodioxan.
The terms "halo" and "halogen" as used herein to identify
substituent moieties, represent fluorine, chlorine, bromine or
iodine, preferably chlorine or fluorine.
The term "aryl," when used alone or in combination, refers to an
unsubstituted or optionally substituted monocyclic or bicyclic
aromatic hydrocarbon ring system having 6 to 12 ring carbon atoms.
Preferred are optionally substituted phenyl, 1-naphthyl, or
2-naphthyl groups. The aryl group may optionally be substituted at
one or more substitutable ring positions (generally at no more than
three positions and most often at one or two positions) by one or
more groups independently selected from alkyl (including haloalkyl
(preferably trifluoromethyl and difluoromethyl)), alkenyl, alkynyl,
alkoxy, aryloxy, nitro, hydroxy, amino, mono- and di-substituted
amino, cyano, halo, alkanoyl, aminocarbonyl, carboxylic acid,
carboxylic acid esters, carboxylic acid amide, an optionally
substituted phenyl (optionally substituted by halo, lower alkyl and
lower alkoxy groups), heterocyclo, or heteroaryl. Preferably, the
aryl group is phenyl optionally substituted with up to four and
more usually with one or two groups, preferably selected from lower
alkyl, lower alkoxy, as well as cyano, trifluoromethyl and
halo.
The terms "aralkyl" and "(aryl)alkyl," alone or in combination are
a species of alkyl as defined herein, and particularly refers to an
alkyl group as defined above in which one hydrogen atom is replaced
by an aryl group as defined above, and includes benzyl, and
2-phenylethyl.
The terms "(heterocyclo)alkyl" and "(heteroaryl)alkyl" alone or in
combination can be considered a species of alkyl as defined herein,
and particularly refers to an to an alkyl group as defined above in
which one hydrogen atom is replaced by a heterocyclo group as
defined above, or by a heteroaryl group as defined above.
The term "alkoxycarbonyl" alone or in combination means a radical
of the formula --C(O)-alkoxy, in which alkoxy is as defined
above.
The term "alkylcarbonyloxy" alone or in combination means a radical
of the formula --O-C(O)-alkyl, in which alkyl is as defined
above.
The term "alkoxyalkanoyl" alone or in combination means a radical
of the formula -alkyl-C(O)--O-alkyl-.
The term "carboxyalkyl" alone or in combination means a radical of
the formula -alkyl-C(O)--OH.
The term "substituted amino" embraces both mono and di-substituted
amino. These terms, alone, or in combination, mean a radical of the
formula --NR'R", where, in the case of mono-substitution, one of R'
and R" is a hydrogen and the other is selected from alkyl,
cycloalkyl, aryl, heterocyclo, (aryl)alkyl, (heterocyclo)alkyl,
heteroaryl and hetero(aryl)alkyl; in the case of di-substitution,
R' and R" are independently selected from alkyl, cycloalkyl, aryl,
heterocyclo, and heteroaryl, or R' and R" together with the
nitrogen atom to which they are both attached form a three to
eight-membered heterocyclo or heteroaryl radical.
The term "amido" refers to the group (--NH--) and the term
"substituted amido" embraces a radical of the formula (--NR--)
where R' has the meaning above in connection with substituted
amino.
The terms "alkanoylamido," "aroylamido," "heterocyclocarbonylamido"
and "heteroatoylamido" mean groups of the formula R--C(O)--NH--
where R is an alkyl, aryl, heteroaryl or heterocyclo group.
The terms "heteroaroyl" and "heterocyclocarbonyl" when used alone
or in combination means groups of the formula R--C(O)-- where R is
a heteroaryl or heterocyclo group.
Unless otherwise defined, the term "optionally substituted" as used
herein, refers to the substitution of a ring system at one or more
positions with one or more groups selected from: C.sub.1-5 alkyl,
C.sub.1-5 alkoxy, an optionally substituted phenyl, cyano, halo,
trifluoromethyl, C.sub.1-5 alkoxycarbonyl, C.sub.1-5 alkyl
carbonyloxy, mono- and bis-(C.sub.1-5 alkyl)-carboxamide, C.sub.1-5
alkylamido, nitro, and mono- and bis-(C.sub.1-5 alkyl)-amino.
Applicants recognize that there may be some overlap in some of the
definitions of the various radical groups. Specific groups are
mentioned, however, such as (aryl)alkyl, and may be particularly
identified in the claims, in order to emphasize their positive
inclusion in the described subject matter, as not only an optional
substituent.
As used herein, when a particular radical generally understood to
have a single point of attachment to a core structure, such as an
alkyl group, is identified in connection with a structure that must
have two points of attachment in the structural core (such as with
the element L in formula (II)), it is understood that the named
radical, e.g., alkyl, refers to the parent radical with a hydrogen
or a site of unsaturation removed to create the second point of
attachment so as to provide the required structure.
Anionic groups according to the present invention, designated as
"A" herein, include carboxylate, sulfate, sulfonate, sulfinate,
sulfonamides, sulfones, phosphate, phosphonate, phosphinate,
tetrazoles, thiophosphate, pyrophosphate, enolate, or a precursor
group that can be converted to an anionic group, either
spontaneously or enzymatically in cells.
A specific embodiment of this invention is phenylglyoxal, shown
below, although many other compounds that can achieve the desired
effect are readily apparent to those skilled in the art, having
regard for this disclosure: ##STR6##
Two additional categories of specific embodiments, in which an
aliphatic chain is capable of binding to the active site of FTase
by hydrophobic interactions, are defined as follows: ##STR7##
Also, substances with complex substituents might be better mimics
of the famesyl group of the substrate FPP and function more
effectively as inhibitors, as for example, the compounds below:
##STR8##
An additional compound that consists of the general formula
R--(C.dbd.O)--(C.dbd.O)--R', or a masked form of such a group as
denoted elsewhere in this application (e.g. acetal, ketal, etc.),
and is contemplated by this disclosure is the following:
##STR9##
The synthesis of the above compound may be carried out as follows:
##STR10##
An additional compound that consists of the general formula
R--(C.dbd.O)--(C.dbd.O) R', or a masked form of such a group as
denoted elsewhere in this application (e.g. acetal, ketal, etc.),
and is contemplated by this disclosure is the following:
##STR11##
The synthesis of the above compound may be carried out according to
the following scheme: ##STR12##
A further example of the class of compounds of the type
R--(C.dbd.O)--(C.dbd.O)--R', or a masked form of such a group as
denoted elsewhere in this application (e.g. acetal, ketal, etc.),
is exemplified by dehydroascorbic acid 6-palmitate, shown below:
##STR13##
This compound may advantageously be synthesized by oxidation of
ascorbic acid 6-palmitate, as follows: ##STR14##
Other embodiments contemplated include compounds in which the
--(CH.sub.2).sub.14 CH.sub.3 group shown in the structural formula
of the 6-palmitate is replaced by a group R as defined elsewhere
herein. These include other 6-esters of dehydroascorbic acid, such
as dehydroascorbic acid 6-farnesenoate, in which the
--(CH.sub.2).sub.14 CH.sub.3 group shown in the structural formula
of the 6-palmitate is replaced by the --CH.dbd.C(CH.sub.3)CH.sub.2
CH.sub.2 CH.dbd.C(CH.sub.3)CH.sub.2 CH.sub.2
CH.dbd.C(CH.sub.3).sub.2 group. Dehydroascorbic acid esters
contemplated by this disclosure are inclusive of the 5-isomers as
well as the 6-isomers.
Some cancer cells in which farnesylation of Ras is blocked employ
the related prenylation reaction geranylgeranylation to attach a
hydrophobic group to Ras to accomplish membrane localization and
continued cancerous behavior of the cell. The enzyme that attaches
the geranylgeranyl group to Ras protein to facilitate the latter's
localization at the inner surface of the cancer cell membrane is
geranylgeranyl protein transferase, also known as protein
geranylgeranyltransferase (herein referred to as GGTase). The
geranylgeranyl group becomes attached to Ras by reaction with
geranylgeranyl diphosphate, another natural FTase substrate, also
known as geranylgeranyl pyrophosphate (herein referred to as GGPP).
In other words, GGTase catalyzes the following reaction, in which
Ras becomes attached to the geranylgeranyl group by displacement of
pyrophosphate (PP.sub.i): ##STR15##
The newly formed geranylgeranyl-Ras localizes at the inner surface
of the cancer cell membrane and causes the cancer cell to divide
without restraint. Thus, a key target in a strategy to retard
cancer cell proliferation is the enzyme GGTase. By reducing or
destroying GGTase activity, either in combination with inactivation
of Ras farnesylation or independently, we hope to prevent Ras
geranylgeranylation, which in turn should prevent localization of
Ras at the inner surface of the cancer cell membrane, thereby
preventing Ras from causing the cancer cells to divide and
proliferate.
Geranylgeranylation of Ras results in the loss from GGPP of
pyrophosphate, which is anionic, a process that might be
facilitated by a chemical group in GGTase's active site that would
favorably interact with pyrophosphate, such as by ionic bonding to
a positively charged group like the side chain of arginine
[R--NH.dbd.C(NH.sub.2).sub.2.sup.+ ]. Thus, chemical reagents that
react with the arginine side chain, such as those that possess the
functional group consisting of two adjacent carbonyl groups, i.e.,
O.dbd.CC.dbd.O, might effectively prevent one or more arginine
residues in GGTase from assisting in the pyrophosphate departure,
thereby blocking or impeding geranylgeranylation of Ras.
Furthermore, to target better the substance to GGTase in preference
to other enzymes, and to target better the substance to the active
site of GGTase in preference to the external or noncatalytic
portion of the enzyme, a preferred embodiment of the invention
would consist of a substance with the dicarbonyl functional group
and other structural features that would direct or otherwise favor
the binding of the agent to the active site of GGTase.
To fine-tune the inactivator to fit the active site of FTase or
GGTase, variation of the distance between the arginine-binding end
and the farnesyl-mimicking or geranylgeranyl-mimicking end can be
achieved through alteration of the length of the spacer, i.e., by
variation of "n" from 0 to 10 in the generalized diagram below:
##STR16##
Additional inactivators incorporating aromatic groups for enhanced
binding to the hydrophobic binding site of FTase or GGTase are
exemplified by the following compound, although there are numerous
possible variants of this type of compound (e.g., the location of
the aromatic ring and the pattern of substitution as ortho, meta,
or para; a heterocyclic ring; a nonaromatic ring; multiple rings;
etc.): ##STR17##
A further embodiment of this invention is based on the observation
that there are two arginine residues in the active site of FTase.
Because such arginine residues are at a characteristic distance
unique to FTase, selective reaction with FTase over other enzymes
and proteins can be accomplished by use of a substance in which two
dicarbonyl functionalities are separated by a spacer, such
substances being represented by the following generalized
formula:
in which D is a dicarbonyl functional group, O.dbd.C--C.dbd.O, or a
masked form of such a group as denoted elsewhere in this
application (e.g. acetal, ketal, etc.), and L is a linker group
that is compatible with and/or has an affinity for the prenylation
enzyme. One embodiment of this invention, in which two dicarbonyl
functional groups are connected via a linker, is
4,4'-biphenyldiglyoxaldehyde, shown below: ##STR18##
This category of compounds is further exemplified by the
bis(.alpha.:-oxoaldehyde) below: ##STR19##
The spacer, (CH.sub.2).sub.n, can be varied in length (for example,
n=10, 11, 1-10, or 1-15) and in other characteristics (e.g., steric
bulk, etc.) to optimize the selectivity for FTase and the ability
to block the FTase active site. Alternatives to the
bis(.alpha.-oxoaldehyde) above include substances of the
bis(.alpha.-oxoketone) type or compounds with both an
.alpha.-oxoketone and (.alpha.-oxoaldehyde functionality, as well
as others readily apparent to those skilled in the art, having
regard for this disclosure. Thus, reaction of FTase with compounds
such as the bis(dicarbonyl) compound shown above might occur with
high efficiency and selectivity, and the covalent attachment of
such compounds to FTase might be particularly effective at blocking
the active site from utilizing the natural substrates FPP and Ras
protein, which need to bind to FTase in the vicinity of the two
arginine residues (for example, in a competitive binding
assay).
Another embodiment of the invention utilizes the characteristics of
the C-terminus of the Ras protein, i.e., the negative charge, for
noncovalent binding to one of the arginine residues and the
dicarbonyl functionality for covalent binding to the other arginine
residue. The theory has been put forth that one of the arginine
residues holds the Ras protein in the active site through
electrostatic binding of the arginine residue's positive charge to
the carboxyl terminus of Ras protein, which is negatively charged.
The other arginine residue binds FPP through the electrostatic
attraction of the negative charge of FPP to the positive charge of
the arginine side chain. Thus, one such class of compounds that
interfere with protein prenylation that is contemplated by this
disclosure consists of four-part molecules symbolized as
follows:
in which each part is defined as follows: A is (1) an anionic
group, e.g., carboxylate, sulfate, sulfonate, sulfinate, phosphate,
phosphonate, thiophosphate, pyrophosphate, enolate, sulfonamide,
sulfone, or tetrazole, (2) a group that can become anionic at
physiological pH, e.g. CO.sub.2 H by deprotonation, or (3) a group
that can be acted upon by an enzyme to reveal a group that can
become anionic at physiological pH, e.g. the ester R'CO.sub.2 R is
hydrolyzed by endogenous esterases to form the acid R'CO.sub.2 H,
which subsequently ionizes at physiological pH to R'CO.sub.2.sup.-
; L is a linker group that is compatible with and/or has an
affinity for the prenylation enzyme, for example an alkyl, alkenyl,
alkynyl, or aryl group. D is a dicarbonyl functional group
(O.dbd.C--C.dbd.O) or a masked form of such a group as denoted
elsewhere in this application (e.g., acetal, ketal, etc.). R is a
group selected from the group consisting of an alkyl, alkenyl,
alkynyl, or aryl group.
Specific embodiments of such a four-part molecule are the following
camphorquinone sulfonamides: ##STR20##
in which n is advantageously equal to 4, 5, 6, 7, 10, 11, 1-10, or
1-15, or another value for optimal binding to prenyltransferases.
It will be apparent to those skilled in the art and having regard
for this disclosure that other linking groups L, other
anion-generating or anionic groups A, and other dicarbonyl groups
D, or masked forms of such groups as denoted elsewhere in this
application (e.g. acetals, ketals, etc.), can be advantageously
substituted for the polymethylene chain --(CH.sub.2).sub.n --,
carboxyl group, and camphorquinone moiety, respectively, in the
above structure. For example, a prodrug, i.e., a substance that has
advantages of stability, solubility, cell permeability, etc., but
that liberates the active or a more active substance in the living
tissues, e.g. spontaneously and/or by action of endogenous enzymes,
light, and/or heat, could advantageously be formulated to be the
camphorquinone sulfonamide esters represented by the structure
shown below: ##STR21##
in which R may be any of a very wide variety of groups, such as
simple straight-chain, branched, or cyclic and heterocyclic
aliphatic groups (including functional groups containing
heteroatoms, such as ether linkages, amino and hydroxyl groups),
and aromatic groups, including heterocycles.
Both of the enantiomeric camphorquinone sulfonamides (i.e., both
(+) and (-) stereoisomers) are contemplated by this patent and
might be advantageously used for interference with protein
prenylation.
The synthesis of camphorquinone sulfonamides can be carried out as
illustrated below: ##STR22##
Additionally, the compound below, when in its ionized form (the
predominant form present at physiological pH), has the
characteristics required for the noncovalent binding of the
carboxylate terminus to one arginine residue, and the covalent
binding of the dicarbonyl functionality to the other arginine
residue: ##STR23##
When m=9, the compound is 11,12-dioxododecanoic acid, a preferred
compound of the invention. Typically m=0 or 1 to 25, 1-10, 1-15,
10, or 11, more preferably 5-13.
Other embodiments can combine the carboxylic acid group and an
.alpha.-oxoaldehyde or, alternatively, a carboxylic acid group and
an (.alpha.-oxoketone group within the same molecule, or other
combinations that are readily apparent to those skilled in the art,
having regard for this disclosure. By modification of the linker
group's structure (e.g., length, steric bulk, etc.), the
specificity of the compound for FTase and the effectiveness of the
compound against FTase can be fine-tuned. Use of known non-covalent
inhibitors of FTase and GGTase as linker or R groups is preferred
to increase specificity for the enzyme active site.
For example, the tetrapeptide portion of Ras that is recognized by
FTase (referred to as CAAX, in which C represents cysteine, A
represents an aliphatic amino acid, and X represents a limited
subset of the naturally occurring amino acids, of which serine is
an example) can be utilized in combination with a dicarbonyl
functionality to target the inactivator to its target, FTase. Such
a compound might be a useful inactivator. It might, however, be
hydrolyzed by cellular proteases, so a peptidomimetic analogue less
prone to cellular hydrolysis might show superior lifetime in a
cell, thereby enhancing its chances of combining with FTase and
inactivating it. An embodiment of a peptidomimetic analogue of the
tetrapeptide in which a dicarbonyl functionality is present is
shown below: ##STR24##
In the structure above, R.sub.1 is a substituent suitable for
recognition and binding to FTase, and R.sub.2 is typically H or
CH.sub.3, for reaction with an arginine residue in the FTase active
site. Many other embodiments of this type are readily apparent to
those skilled in the art, having regard for this disclosure.
There are numerous ways to synthesize .alpha.-dicarbonyl compounds.
One is by oxidation of a monocarbonyl compound with selenium
dioxide (SeO.sub.2), shown below: ##STR25##
Another method of preparation of .alpha.-dicarbonyl compounds is by
alkylation of 2,3-butanedione, illustrated below: ##STR26##
Another means of synthesis of .alpha.-dicarbonyl compounds is
illustrated below, starting with citronellal: ##STR27##
Other embodiments of the invention are substances in which one or
more carbonyl groups are masked as a hydrate [C(OH).sub.2 ], a
hemiacetal or hemiketal [C(OH)(OR")], an acetal or a ketal
[C(OR")(OR'")], an acylal or related compound
[C(OC(.dbd.O)R")(OC(.dbd.O)R'")], a bisulfite addition compound
[C(OH)(SO.sub.3.sup.-)], an enol (C.dbd.COH), an enol ether
(C.dbd.COR"), an enol ester [C.dbd.COC(.dbd.O)R"], and so forth,
wherein R" and R'", which may be the same or different, are alkyl,
alkenyl, and/or aryl groups. Such masked carbonyl groups may
produce the carbonyl form of the reagent in solution. Acetals and
ketals with hydrophobic side chains, including those that resemble
the farnesyl group, are shown below: ##STR28##
The examples below show that FTase is sensitive to inactivation by
phenylglyoxal and by 2-oxododecanal, both of which are
.alpha.-dicarbonyl compounds. New opportunities exist for the
development of dicarbonyl compounds and related substances that
might prove to be inactivators of prenylation enzymes with varying
potencies and specificities. A specific protein designated K-Ras is
the most common mutant Ras protein found in human cancers.
Farnesylation inhibitors, however, can not prevent cellular
prenylation of K-Ras because geranylgeranylation of K-Ras takes
place. If GGase has an active site arginine, -dicarbonyl compounds
like PGO might likewise be effective inactivators of that enzyme as
well.
The ability of the .alpha.-dicarbonyl functional group to achieve
FTase inactivation is described in the examples below, and
compounds of this type offer a new approach to the treatment and
control of cancer. Suitable levels of enzyme activity reduction
range from at least 10% to at least 75% or 95% or even 100%. Any
level can be selected as desired, using appropriate chemical
substituents to achieve at least 10, 20, 30, 40, 50, 60, 70, 75,
80, or 90% reduction in enzyme activity. Typical routes of
administration of these substances that might be suitable for
clinical applications are oral and intravenous, although other
routes might prove beneficial in specific cancers (e.g.,
intraperitoneal, intramuscular, subcutaneous, intrathecal, or
topical for skin cancers). Doses and frequency of administration
are to be selected based upon considerations of beneficial effects
(e.g., tumor growth reduction or tumor shrinkage) compared to side
effects (e.g., systemic toxicity). Combination with other
anticancer agents to produce synergistic effects of benefit to the
patient are also possible. Combination therapy might be based on
two strategies. One is to interfere with different biochemical
processes to increase tumor cell killing. Another is to hamper
development of drug resistance, which is less likely to occur
simultaneously in tumor cells exposed to anticancer agents based on
interference with different biochemical pathways in the tumor
cells.
Compounds according to the invention may be purified free of
impurities, preferably to a level of at least 50, 70, 90, or 95%.
The compounds may be formulated in suitable pharmaceutically
acceptable diluents, excipients or carriers. These can provide for
additional properties, such as enhanced absorption, slow release,
tissue or organ targeting, etc.
In summary an improved method of interference with protein
prenylation in tumor cells has been described that may prevent or
hamper the proliferation of tumor cells, possibly resulting in a
decrease in tumor size and/or disappearance of the cancer.
EXAMPLE 1
An example of an embodiment of this invention is phenylglyoxal
monohydrate, shown below, which is in equilibrium with the
dicarbonyl form: ##STR29##
The application of phenylglyoxal monohydrate to solutions of FTase
resulted in the rapid and virtually complete inactivation of the
enzyme in a matter of seconds to minutes, depending upon the
concentration of phenylglyoxal monohydrate applied. For example,
the catalytic ability of FTase was monitored by an assay procedure
in which FTase transferred the farnesyl group from FPP to a peptide
substitute for Ras. The peptide substitute for Ras in this assay
had structural elements in common with Ras, in particular a
sequence of four amino acids cysteinyl-valy-lysyl-serine
(cys-val-lys-ser). In addition the peptide contained an amino acid
residue that had a fluorescent marker attached, dansyl glycine
(DNS-gly). Farnesyl group transfer by FTase from FPP to
DNS-gly-cys-val-lys-ser resulted in an increase in fluorescence due
to the attachment of the hydrophobic group to the cysteine near the
dansyl group. Thus, enzyme activity is revealed by an increase in
fluorescence of the dansyl group, as shown in the accompanying FIG.
1.
Inactivation of the enzyme FTase occurred when the FTase was
preincubated with phenylglyoxal monohydrate, as evidenced by (1) a
decrease in enzyme activity with increasing time of preincubation
with phenylglyoxal monohydrate; and (2) a decrease in enzyme
activity with increasing concentration of phenylglyoxal
reumonohydrate. Because of interference of phenylglyoxal
monohydrate with the fluorescence assay used, phenylglyoxal
monohydrate was removed from the FTase preincubation mixture by
ultrafiltration prior to the assay being performed. The
accompanying FIG. 2 shows the loss of activity of FTase due to
inactivation by phenylglyoxal monohydrate.
Evidence for the binding of phenylglyoxal in the farnesyl
pyrophosphate binding region of the enzyme was obtained by
preincubation of the enzyme with farnesyl pyrophosphate, followed
by treatment with phenylglyoxal. As shown in the accompanying FIG.
3, higher concentrations of farnesyl pyrophosphate more effectively
reduced the inactivation of FTase by phenylglyoxal. This suggests
that the farnesyl pyrophosphate bound to and blocked phenylglyoxal
from the site of the reaction that brings about inactivation,
namely chemical modification of an active-site arginine side
chain.
A detailed description of the results summarized above follows.
Upon farnesylation by protein farnesyltransferase (FTase), .sup.1
Ras protein becomes localized at the inner surface of the cell
membrane. Localization is essential for the mitogenic role of
mutant Ras protein, which acts as a switch for cancer cell
proliferation. We found that phenylglyoxal (PGO), which is a
protein-modification reagent that is specific for arginine,
potently obstructs farnesylation of a Ras model peptide by FTase.
Covalent modification interferes with catalysis and results in
mechanism-based inactivation of FTase. FTase activity in vitro was
monitored by a cell free fluorescence assay. FTase was rapidly
inactivated by preincubation with PGO. At 20 mM PGO, FTase activity
was totally lost within 2 minutes. Inactivation rates at 10 mM PGO
and 5 mM PGO were successively slower. Preincubation of FTase with
FPP prior to incubation with PGO decreased the ability of PGO to
inactivate FTase. These results imply that a bimolecular reaction
occurred between PGO and FTase, probably in the vicinity of the FPP
binding site, which resulted in the inactivation of the enzyme.
These findings reveal the value of -dicarbonyl compounds for
mechanistic studies of FTase and for potentially selective
regulation of protein farnesylation for possible anticancer
chemotherapy.
Posttranslational modification of a small number of proteins is
carried out by protein farnesyltransferase (FTase), which attaches
the 15-carbon farnesyl group of farnesyl pyrophosphate to the
sulfhydryl group of a cysteine residue near the C-terminus of the
substrate protein. Noteworthy among the proteins that are substrate
for FTase is Ras protein, which upon farnesylation becomes
localized at the lipid bilayer of the inner surface of the cell
membrane. Localization is essential for the mitogenic role of Ras,
which acts as a switch for cell division. Cycling between the
inactive form (GDP-bound Ras) and the active form (GTP-bound Ras),
is part of the normal mechanism of cell growth regulation.
Mutant forms of Ras protein have lost GTPase activity and trigger
unrestrained cell division. Approximately one third of all human
cancers exhibit a mutant ras genotype, with substantial variations
in the incidence from tumor type to tumor type. This has made
blockage of farnesylation of mutant Ras protein an active area of
research as it appears that inhibition of both normal and mutant
Ras protein farnesylation is more harmful to cancer cells than
normal cells. The challenges to this coup de main against cancer,
however, are manifold. Some cancer cell lines with mutant ras
genotypes are insensitive to FTase inhibitors, and some cancer cell
lines without mutant ras genotypes are sensitive to FTase
inhibitors. This may mean that a delicate balance between reduction
in farnesylation of different proteins is required for effective
anticancer strategies. A possible addition to the armamentarium of
farnesylation regulators based on a new approach is described in
this report.
Recognition of Ras and related protein substrates of FTase is based
on a structural motif consisting of a C-terminal CAAX sequence, in
which A represents an aliphatic amino acid and X is preferentially
serine. The related posttranslational geranylgeranylation, in which
the 20-carbon geranylgeranyl chain is attached to the cysteine of
CAAX by geranylgeranyltransferase (GGTase), occurs preferentially
when X is methionine.
Nucleophilic attack by the cysteine sulfhydryl group of CAAX,
activated by an enzyme-bound zinc ion, results in Ras
farnesylation, with departure of pyrophosphate from FPP. We
surmised that catalysis of farnesylation by FTase might be
facilitated by salt-bridge formation (i.e., electrostatic
stabilization) between the pyrophosphate leaving group of famesyl
pyrophosphate and a cationic amino acid side chain such as the
guanidinium group of arginine. This speculation was strengthened by
the recent publication of some aspects of FTase structure obtained
from X-ray crystallographic analyses. An arginine (Arg.sup.291) was
found to be in close proximity to the binding site of the
diphosphate portion of FPP and the active-site zinc ion, near the
upper part of the barrel-shaped active site. Another key arginine
in the bottom of the active site, Arg.sup.202, is proposed to form
a salt bridge with the carboxyl group of the CAAX terminus of the
Ras peptide substrate. In addition the aliphatic part of the
arginine side chain is buttressed with the FPP aliphatic chain.
FTase's preference for FPP relative to GGPP can be understood
because the latter would project from the cleft and be poorly
positioned for transfer of the geranylgeranyl group to the protein
substrates of FTase.
We report herein that a protein-modification agent, phenylglyoxal,
that is specific for the arginine side chain potently obstructs
farnesylation of a Ras model peptide by FTase. This approach of
specific covalent modification of FTase to interfere with catalysis
results in mechanism-based inactivation of FTase, in contrast to
the numerous noncovalent inhibitors that have been identified.
Covalent modification is in principle virtually irreversible and
might be tailored to active site residues of FTase. Inactivation
thus opens the door to mechanistic studies and potentially
selective obstruction of FTase and/or GGTase for possible
anticancer chemotherapeutic benefit.
Experimental Procedures:
Materials. All reagents were from Sigma Chemical Co. and used
without further purification, except as noted.
Purification of PGO-PGO (Aldrich Chemical Co.) was dissolved in
boiling water that contained charcoal, and the mixture was gravity
filtered through Celite while hot. Upon cooling, the solution was
filtered by suction to obtain the PGO, and the PGO was repeatedly
recrystallized from water until its .sup.1 H NMR spectrum and
melting point indicated the absence of impurities, such as
polymeric material. All PGO solutions were freshly prepared before
use in the studies described below.
Preparation of Ds-GCVLS Ds-GCVLS was prepared by a method analogous
to a published procedure. Synthesis was performed by use of a
Millipore 9050 Plus PepSynthesizer that employed standard Fmoc
solid phase peptide synthesis methodology. PS-PEG resin and Fmoc
protected amino acids were obtained from Millipore. The
N-dansylglycine was from Sigma Chemical Co. Peptide purification
was performed by RP-HPLC on an Alitech Macrosphere RP300 C8 column.
Elution was accomplished with a linear gradient from aqueous 10 mM
trifluoroacetic acid to 10 mM trifluoroacetic acid in acetonitrile.
Lyophilization yielded a pale yellow solid. Stock solutions were
prepared in 50 mM Tris containing 5 mM DTT and 0.2%
n-octyl-.beta.-D-glucopyranoside (Anatrace, Inc.), pH 7.7.
Concentrations of Ds-GCVLS were determined by use of the extinction
coefficient of the dansyl group at 340 nm (=4250 M.sup.-1 cm.sup.-
1). Stock solutions were stored at -20 C. FTase (stored at
-70.degree. C.) was a generous gift from Professor Patrick Casey at
Duke University.
FTase assay The fluorescence assay conditions were adapted from a
published method. The FTase assay consisted of the measurement of
the rate of FTase-catalyzed farnesylation of the fluorescent
pentapeptide Ds-GCVLS. Upon farnesylation of the cysteine, the
fluorescence emission at=505 nm of the dansyl group is enhanced due
to the proximity of the hydrophobic isoprenoid group. Enzyme
activity (approximately 60 nM FTase) was monitored at saturating
substrate concentrations (10 M farnesyl pyrophosphate, ammonium
salt, prepared by successive dilutions of a commercial 2.3 mM
solution in methanol: 10 mM aqueous NH.sub.4 OH (7:3) into assay
buffer, and 1.0 M Ds-GCVLS) by measurement of the increase in
fluorescence emission observed at=505 nm as FTase farnesylated
Ds-GCVLS for a period of 10 min. The assay buffer was selected for
unreactivity toward PGO: 50 mM NaHEPES, 5 mM DTT, 5 mM MgCl.sub.2,
10 .mu.M ZnCl.sub.2, and 0.2% n-octyl-.beta.-D-glucopyranoside, pH
7.5. Fluorescence emission was measured with a JASCO FP-77
spectrofluorimeter fitted with a temperature-controlled cuvette
holder connected to a 30 C constant temperature bath. All
fluorescence assays were conducted in a 4 mm 4 mm quartz cuvette
with excitation at 340 nm.
Inactivation of FTase by PGO FTase was incubated at 30 C in the
presence and absence of PGO at various concentrations in 50 mM
NaHEPES, 5 mM MgCl.sub.2, 10 .mu.M ZnCl.sub.2, and 0.2%
n-octyl-.beta.-D-glucopyranoside, pH 7.5 (i.e., assay buffer
without DTT). Periodically, aliquots of the FTase-PGO reaction
mixture were removed and assayed for FTase activity. Because of
interference of PGO with the fluorometric assay, the aliquot was
first subjected to solvent exchange by size exclusion membrane
concentration to remove PGO. The solvent exchange utilized Microcon
(Millipore Co.) 30 kD molecular weight cutoff microconcentrators.
This also served to stop further inactivation of the enzyme by PGO.
A 100-L aliquot of FTase-PGO reaction mixture was pipetted into a
Microcon concentrator, which was centrifuged at 12,000 RPM for 8
min at 4 C. The concentrate was resuspended in 100 L of assay
buffer, and centrifugation was repeated. The final concentrate was
resuspended in 90 L of assay buffer, and the resulting solution was
recovered by inversion of the concentrator and centrifugation at
3500 RPM for 3 min at 4 C. The solution was then assayed for FTase
activity as described above. A control was handled as above, except
that PGO was omitted from the incubation mixture.
FPP protection of FTase against inactivation by PGO FTase was
treated with FPP at various concentrations in assay buffer without
DTT for 2 min at 30.degree. C. prior to incubation of the mixture
with 10 mM PGO for 30 min at 30.degree. C. The control was
incubated with FPP at the corresponding concentration for the full
length of time, but without PGO. The mixtures were subsequently
subjected to microconcentration and assay, as described above.
Results and Discussion:
FTase activity in vitro was monitored by a fluorescence assay,
shown in FIG. 1, in which activity was determined from the initial
slope of a plot of the fluorescence increase vs. time. FTase was
rapidly inactivated by preincubation with PGO, as shown in FIG. 2.
At 20 mM PGO, FTase activity was totally lost before the first
assay at 2 minutes after mixing of PGO and FTase (data not shown).
Inactivation by 10 mM PGO was slower but still quite rapid. At 5 mM
PGO the rate of inactivation was reduced. These results imply that
a bimolecular reaction occurred between PGO and FTase, which
resulted in the inactivation of the enzyme.
Inactivation kinetics differ from inhibition kinetics. Inactivation
kinetics typically consist of the bimolecular conversion of the
active form of the enzyme (E) into impaired and/or completely
inactive form(s) of the enzyme (E'), as shown in the equation
below, where PGO is the inactivator: ##STR30##
Because the inactivator is typically present in large excess over
the enzyme and therefore remains at essentially constant
concentration, disappearance of enzyme activity follows
pseudo-first-order kinetics, described by the following
equation:
in which [E].sub.t is the enzyme concentration or activity
remaining at time t, [E].sub.0 is the initial enzyme concentration
or activity, and [PGO] is the concentration of PGO. Thus, a plot of
log [E].sub.t /[E].sub.0, i.e., log (fraction of activity
remaining), versus time is expected to be linear, with a slope of
k[PGO]/2.303. Thus, the time-dependence and
inhibitor-concentration-dependence of loss of enzyme activity are
indicative of inactivation of the enzyme.
Previously, the stepwise formation of a 2:1 PGO-enzyme adduct has
been seen kinetically as a biphasic time course of inactivation of
porcine liver prenyltransferase, presumably due to rapid,
reversible 1:1 adduct formation resulting in reduced activity of
the enzyme, followed by slower, irreversible 2:1 adduct formation,
and conversion of the enzyme into a less active form. Some
indication of similar behavior is seen in FIG. 2, especially at low
PGO concentration.
Preincubation of FTase with FPP prior to incubation with PGO
decreased the ability of PGO to inactivate FTase, as shown in FIG.
3. This implies that an active site arginine residue is the
principal site of the reaction with PGO that inactivates the
enzyme. When arginine modification occurs in the FPP binding site,
then obstruction of subsequent binding of FPP would be expected to
prevent farnesylation of the substrate pentapeptide. The low
concentration of PGO required for inactivation of the enzyme is
also consistent with the idea that specific modification of an
active-site arginine has occurred.
It is not possible to rule out that the arginine side-chain
modification results in destruction of the catalytic ability of the
active-site arginine without blockage of FPP binding. Likewise,
some modification of lysine side chains and -amino groups is
possible with phenylglyoxal, although under the short incubation
times used in these studies, such modification would be expected to
be of minor importance. Destruction of enzyme activity by
modification of numerous arginines, which might for example lead to
gross conformational changes and concomitant loss of catalytic
ability, also would not be expected to occur under these
conditions. In either case, the observed protective effect of FPP
would not necessarily have been expected. Future studies, however,
are required to establish whether Arg.sup.291 is the principal site
of modification of FTase by PGO. Such studies will also ascertain
whether another amino acid is a significant target of PGO
modification.
EXAMPLE 2
In the same manner as Example 1, compound 2-oxododecanal was tested
for inactivation of FTase in the fluorescence assay system
described, except as noted below. Final concentrations in the
incubation mixture were 5, 10, and 35 mM 2-oxododecanal. A stock
solution of 2-oxododecanal dissolved in dimethyl sulfoxide (DMSO)
was employed. The final concentration of DMSO in the incubation
mixture (as well as the control) was 5%. The results are shown in
FIG. 4. It was found that 2-oxododecanal rapidly inactivated FTase,
probably by reaction with one or both of the active site arginine
residues. This would block access of one or both of the substrates
(FPP and/or the peptide substitute for Ras protein) and/or block
the catalytic function of one or both of the arginine residues,
resulting in inactivation of the enzyme.
EXAMPLE 3
In the same manner as EXAMPLE 1, compound
5,9-dimethyl-8-decene-2,3-dione was tested for inactivation of
FTase in the fluorescence assay system described, except as noted
below. A stock solution of 5,9-dimethyl-8-decene-2,3-dione in 5%
DMSO was employed. The final concentration of DMSO in the
incubation mixture (as well as the control) was 4.6%. FTase was at
15 nM, and the Microcon concentration step was omitted. It was
found that 5,9-dimethyl-8-decene-2,3-dione rapidly inactivated
FTase. After 30 minutes of incubation with 17, 68, or 171 .mu.M
5,9-dimethyl-8-decene-2,3-dione, FTase activity was reduced to 38%,
18%, or 16% of the initial control value, respectively.
EXAMPLE 4
In the same manner as EXAMPLE 1, dehydroascorbic acid 6-palmitate
was tested for inactivation of FTase in the fluorescence assay
system described, except as noted below. A stock solution of
dehydroascorbic acid 6-palmitate in 2% DMSO in 5 mM sodium acetate,
pH 3.9, was employed. The final concentration of DMSO in the
incubation mixture (as well as the control) was 0.46%. FTase was at
15 nM, and the Microcon concentration step was omitted. It was
found that dehydroascorbic acid 6-palmitate rapidly inactivated
FTase. After 30 minutes of incubation with 7, 28, or 141 .mu.M
dehydroascorbic acid 6-palmitate, FTase activity was reduced to
75%, 34%, or 22% of the initial control value, respectively.
EXAMPLE 5
In the same manner as EXAMPLE 1, compound
4,4'-biphenyldiglyoxaldehyde was tested for inactivation of FTase
in the fluorescence assay system described, except as noted below.
A stock solution of 4,4'-biphenyldiglyoxaldehyde in 5% DMSO was
employed. The final concentration of DMSO in the incubation mixture
(as well as the control) was 4.6%, and FTase was at 15 nM. It was
found that 4,4'-biphenyldiglyoxaldehyde rapidly inactivated FTase.
After 30 minutes of incubation with 23 or 93 .mu.M
4,4'-biphenyldiglyoxaldehyde, FTase activity was reduced to 68% or
11% of the initial control value, respectively.
EXAMPLE 6
As a test of the importance of the presence of a dicarbonyl
functional group, a compound analogous to that used in EXAMPLE 5
but without such a group was tested. Thus, in the same manner as
EXAMPLE 1, compound 4,4'-diacetylbiphenyl, whose structure is as
follows: ##STR31##
was tested for inactivation of FTase in the fluorescence assay
system described, except as noted below. A stock solution of
4,4'-diacetylbiphenyl in 5% DMSO was employed. The final
concentration of DMSO in the incubation mixture (as well as the
control) was 4.6%, FTase was at 15 nM. It was found that
4,4'-diacetylbiphenyl did not inactivated FTase. Even after 120
minutes of incubation with 23 JM 4,4'-diacetylbiphenyl, FTase
activity was the same as the control, within experimental
error.
EXAMPLE 7
In the same manner as EXAMPLE 1, compound 2,3-pentanedione,
CH.sub.3 CH.sub.2 (C.dbd.O)--(C.dbd.O)CH.sub.3, was tested for
inactivation of FTase in the fluorescence assay system described,
except as noted below. The assay was carried out in the presence of
50 mM sodium borate, pH 7.5. FTase was at 15 mM, and the Microcon
concentration step was omitted. It was found that 2,3-pentanedione
rapidly inactivated FTase. After approximately 30 minutes of
incubation with 17, 33, or 66 mM 2,3-pentanedione, FTase activity
was reduced to 69%, 55%, or 37% of the initial control value,
respectively.
Footnotes: The abbreviations used are: Ds-GCVLS, the dansyl-labeled
pentapeptide
N-dimethylaminonaphthalenesulfonyl-Gly-Cys-Val-Leu-Ser; DTT,
dithiothreitol; Fmoc, 9-fluorenylmethoxycarbonyl; FTase, human
recombinant protein farnesyltransferase; FPP, farnesyl
pyrophosphate; GGPP, geranylgeranyl pyrophosphate; GGTase, protein
geranylgeranyltransferase; HEPES,
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; PGO,
phenylglyoxal monohydrate; Tris,
tris(hydroxymethyl)aminomethane.
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